The invention relates to a method of transmission electron microscopy imaging of samples arranged on an electron microscopy grid. Furthermore, the invention relates to a transmission electron microscope, which is configured for transmission electron microscopy imaging of sample arrays. Applications of the invention are available in the imaging of multiple samples, including e. g. biological materials, like cellular macromolecules, crystals thereof, cellular components, or other biological particles, e. g. viruses, or non-biological materials, like nanoparticles.
Transmission electron microscopy (TEM) is a widely used method for imaging samples with an imaging resolution in the sub-nm range. The sample to be imaged is arranged on an electron microscopy grid, which typically comprises a circular mesh of metallic filaments covered with an electron transparent film, e. g. carbon film. Using a transfer device, the grid is loaded into an evacuated microscope column, wherein an imaging electron beam is directed through the sample and a transmission image is collected with a detector. Sample handling is a time consuming procedure, which represents a serious restriction if large quantities of samples are to be imaged, e. g. for high throughput imaging of biological samples.
In order to improve the TEM imaging of large quantities of samples, the creation of a sample array on the grid has been proposed in WO 2013/109405 A1. The sample array is deposited using an inkjet dispenser ejecting small droplets on the grid surface. A drive mechanism is used for positioning the grid relative to the inkjet dispenser. In order to remove excess liquid, which may comprise sample liquid or a liquid staining substance, the grid surface is provided with blotting material. The blotting material comprises e. g. porous membranes, which are capable of sucking liquids on the grid surface.
Due to the following disadvantages, the technique of WO 2013/109405 A1 has practical limitations. Firstly, the conventional grid handling requires a complex mechanical structure, e. g. for adjusting a grid holder relative to the inkjet dispenser. Loading the transfer device with the sample array grid may degrade the sample array, e. g. by an unintended bending of the grid. Furthermore, the conventional technique has a disadvantage in terms of a limited reliability of registering the samples for traceability. If the orientation of the sample array grid is changed during the loading to the transfer device, the sample identification can be lost.
The application of multiple biological samples on a single TEM grid also has been described by P. Castro-Hartmann et al. in “Ultramicroscopy”, vol. 135, 2013, p. 105-112. The samples are deposited on the grid surface with a contact needle printer, followed by washing, staining and blotting steps. As a disadvantage of this technique, the contact needle printer requires TEM grids with high mechanical strength.
Providing small samples on a TEM grid without need for a blotting step is proposed in US 2011/0238225 A1. A liquid droplet sample is disposed with a first capillary on the grid surface, and excess liquid is removed from the droplet with a second capillary. Although this technique does not require blotting material on the grid surface, the conventional application of the capillaries results in limitations in terms of mechanically holding and precise position control of the capillaries. Furthermore, a sample identification for positive traceability is not possible with the device of US 2011/0238225 A1. Accordingly, this conventional technique is not applicable for high-throughput tasks, e. g. for removing liquid from a sample array on a TEM grid.
It is a first objective of the invention to provide an improved method of electron microscopy imaging of samples, being capable of avoiding disadvantages or limitations of conventional techniques. In particular, the disadvantages in terms of a cumbersome handling of sensitive grids, complex measures for blotting steps and/or limited registration reliability are to be avoided. It is another objective of the invention to provide an improved electron microscope, being capable of avoiding disadvantages of conventional electron microscopes and in particular facilitating the handling of grids with sample arrays.
These objectives are solved with a method and an electron microscope comprising the features of the invention.